Morphological mutants of filamentous fungi

ABSTRACT

The present invention relates to methods of obtaining a mutant cell from a filamentous fungal parent cell, comprising: (a) obtaining mutant cells of the parent cell; (b) identifying the mutant cell which exhibits a more restricted colonial phenotype and/or a more extensive hyphal branching than the parent cell; and (c) identifying the mutant cell which has an improved property for production of a heterologous polypeptide than the parent cell, when the mutant and parent cells are cultured under the same conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/816,239 filed Mar. 13, 1997, which is a continuation-in-part of U.S.application Ser. No. 08/726,114 filed Oct. 4, 1996, which claimspriority from U.S. provisional application Ser. No. 60/010,238 filedJan. 19, 1996, which applications are fully incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to novel fungi having improvedcapacity for secretion of recombinant polypeptides, and a method forimproving such secretion.

[0004] 2. Description of the Related Art

[0005] Filamentous fungi have become established as widely used hostcell systems for the production of recombinant polypeptides. In manycases, however, fungi which have the desirable traits of ease oftransformability and heterologous polypeptide expression do notnecessarily have the most desirable characteristics for successfulfermentation. For example, growth morphology during fermentation may notbe optimal, since many cultures become quite viscous as biomassincreases. Increased viscosity limits the ability to mix and aerate thefermentation culture, leading to oxygen and nutrient starvation of themycelia, which in turn become inviable and unproductive limiting theyield of the polypeptide of interest. On the other hand, filamentousfungal strains showing good fermentation morphology are not necessarilythe best production strains in terms of quantity of enzyme produced.Therefore, for commercial purposes, there is a need for filamentousfungal hosts which combine the capacity for expression of commercialquantities of recombinant polyp eptide with satisfactory growthcharacteristics, such as rapid growth and low viscosity, therebyenhancing productivity during fermentation.

[0006] Screening of large numbers of mutants for improved fermentationmorphology is quite difficult, and morphological mutant strains haveoften been isolated based on unusual colony morphology on solid medium.Traditionally, morphological mutants have been isolated in transformedstrains that contain multiple copies of a heterologous gene. The mutantsare then analyzed for fermentation growth characteristics andheterologous gene expression. Although this method may be useful inidentifying improved expression strains, the relationship between anyparticular fungal growth morphology and a strain's ability to produce alarge quantity of secreted polypeptide has yet to be established.Morphological mutants are also occasionally recovered in polypeptideexpression improvement screens, following mutagenesis of a transformedstrain, but again, the study of these strains has not led to anysignificant insight into the control of morphology. In addition,morphologically “improved” strains of parental strains containingheterologous gene expression cassettes are not suitable as generalexpression hosts since they cannot be used for the exclusive expressionof other heterologous polypeptides.

[0007] It is an object of the present invention to provide methods forproducing and identifying useful morphological mutants for heterologouspolypeptide production.

SUMMARY OF THE INVENTION

[0008] The present invention relates to methods of obtaining a mutantcell from a filamentous fungal parent cell, comprising: (a) obtainingmutant cells of the parent cell; (b) identifying the mutant cell whichexhibits a more restricted colonial phenotype and/or more extensivehyphal branching than the parent cell; and (c) identifying the mutantcell which has an improved property for production of a heterologouspolypeptide than the parent cell, when the mutant and parent cells arecultured under the same conditions.

[0009] The invention also relates to mutant filamentous fungal cellsproduced by the methods of the present invention.

[0010] The present invention also relates to methods for producing aheterologous polypeptide, comprising: (a) culturing a mutant cell of thepresent invention which comprises a nucleic acid sequence encoding theheterologous polypeptide; and (b) recovering the heterologouspolypeptide.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 shows a restriction map of pJeRS23.

[0012]FIG. 2 shows the colony growth of control strain HowB425 andcolonial mutant JeRS316 on PDA+uridine solid medium.

[0013]FIG. 3 shows a graphic illustration of the distribution of lipaseexpression in HowB425 (control) and JeRS316 (mutant) transformants.

[0014]FIG. 4 shows a graphic illustration of the distribution of lipaseexpression in HowB425 (control) and JeRS317 (mutant) transformants.

[0015]FIG. 5 shows a comparison of the heterologous lipase productivephase of the fermentation of control strain vs. mutant strain.

[0016]FIG. 6 shows a restriction map of pCaHj418.

[0017]FIG. 7 shows a restriction map of pDM148.

[0018]FIG. 8 shows a restriction map of pDM149.

[0019]FIG. 9 shows a restriction map of pJaL154.

[0020]FIG. 10 shows a restriction map of pMT1612.

[0021]FIG. 11 shows a restriction map of pJRoy30.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention relates to methods of obtaining a mutantcell from a filamentous fungal parent cell, comprising: (a) obtainingmutant cells of the parent cell; (b) identifying the mutant cell whichexhibits a more restricted colonial phenotype and/or more extensivehyphal branching than the parent cell; and (c) identifying the mutantcell which has an improved property for production of a heterologouspolypeptide than the parent cell, when the mutant and parent cells arecultured under the same conditions.

[0023] The parent cell may be mutagenized by methods known in the art.For example, mutagenesis of the parent cell can be achieved byirradiation, e.g., UV, X-ray, or gamma radiation of the parent cell.Furthermore, mutagenesis can be obtained by treatment with chemicalmutagens, e.g., nitrous acid, nitrosamines, methyl nitrosoguanidine, andbase analogues such as 5-bromouracil. Most conveniently, the mutagen isapplied to spores of the parent strain, and the surviving spores areplated out for growth on a solid medium. It will also be understood thatmutants can also be naturally occurring variants in a population in theabsence of a specific mutagenesis procedure, either by selection,screening, or a combination of selection and screening. See, forexample, Wiebe et al., 1992, Mycological Research 96: 555-562 and Wiebeet al., 1991, Mycological Research 95: 1284-1288 for isolatingmorphological mutants of Fusarium strain A3/5. Therefore, for purposesof the present invention, the term “mutants” also encompasses naturallyoccurring variants or mutants without deliberate application ofmutagens, i.e., spontaneous mutants.

[0024] The filamentous fungal parent cell may be any filamentous fungalcell. Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a vegetative mycelium composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic.

[0025] In the present invention, the filamentous fungal parent cell maybe a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium,Thielavia, Tolypocladium, and Trichoderma or teleomorphs or synonymsthereof. Known teleomorphs of Aspergillus include Eurotium, Neosartorya,and Emericella. Strains of Aspergillus and teleomorphs thereof arereadily accessible to the public in a number of culture collections,such as the American Type Culture Collection (ATCC), Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau VoorSchimmelcultures (CBS), and Agricultural Research Service Patent CultureCollection, Northern Regional Research Center (NRRL). Known teleomorphsof Fusarium of the section Discolor include Gibberella gordonii,Gibberella cyanea, Gubberella pulicaris, and Gibberella zeae.

[0026] In a preferred embodiment, the filamentous fungal parent cell isan Aspergillus cell. In another preferred embodiment, the filamentousfungal parent cell is an Acremonium cell. In another preferredembodiment, the filamentous fungal parent cell is a Fusarium cell, e.g.,a Fusarium cell of the section Elegans or of the section Discolor. Inanother preferred embodiment, the filamentous fungal parent cell is aHumicola cell. In another preferred embodiment, the filamentous fungalparent cell is a Myceliophthora cell. In another preferred embodiment,the filamentous fungal parent cell is a Mucor cell. In another preferredembodiment, the filamentous fungal parent cell is a Neurospora cell. Inanother preferred embodiment, the filamentous fungal parent cell is aPenicillium cell. In another preferred embodiment, the filamentousfungal parent cell is a Thielavia cell. In another preferred embodiment,the filamentous fungal parent cell is a Tolypocladium cell. In anotherpreferred embodiment, the filamentous fungal parent cell is aTrichoderma cell. In a more preferred embodiment, the filamentous fungalparent cell is an Aspergillus oryzae, Aspergillus niger, Aspergillusfoetidus, Aspergillus nidulans, or Aspergillus japonicus cell. Inanother more preferred embodiment, the filamentous fungal parent cell isa Fusarium strain of the section Discolor (also known as sectionFusarium). For example, the filamentous fungal parent cell may be aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum,Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, orFusarium trichothecioides cell. In another preferred embodiment, thefilamentous fungal parent cell is a Fusarium strain of the sectionElegans, e.g., Fusarium oxysporum. In another more preferred embodiment,the filamentous fungal parent cell is a Humicola insolens or Humicolalanuginosa cell. In another more preferred embodiment, the filamentousfungal parent cell is a Myceliophthora thermophilum cell. In anothermore preferred embodiment, the filamentous fungal parent cell is a Mucormiehei cell. In another more preferred embodiment, the filamentousfungal parent cell is a Neurospora crassa cell. In another morepreferred embodiment, the filamentous fungal parent cell is aPenicillium purpurogenum cell. In another more preferred embodiment, thefilamentous fungal parent cell is a Thielavia terrestris cell. Inanother more preferred embodiment, the Trichoderma cell is a Trichodermareesei, Trichoderma viride, Trichoderma longibrachiatum, Trichodermaharzianum, or Trichoderma koningii cell.

[0027] The mutant cells produced in the first step are then screened forthose mutant cells which (a) exhibit a more restricted colonialphenotype and/or more extensive hyphal branching than the parent cell;and (b) have an improved property for production of a heterologouspolypeptide than the parent cell, when the mutant and parent cells arecultured under the same conditions. In a preferred embodiment, themutant cells are inspected first for the more restricted colonialphenotype and/or more extensive hyphal branching, more preferably, firstfor the more restricted colonial phenotype followed by the moreextensive hyphal branching.

[0028] The mutant cells of the present invention may have a colonialphenotype which is more restricted than the parent cell when the mutantand parent cells are grown on the same solid medium. A mutant cellhaving “more restricted colonial phenotype” is defined herein as amutant cell having a reduced radial extension rate than a parent cellwhen the mutant cell and parent cell are grown on the solid medium.Preferably, the colonial phenotype of the mutant cells is at least about10%, more preferably at least about 20%, and most preferably at leastabout 30% more restricted than the parent cell.

[0029] The mutant cells of the present invention may also have a moreextensive hyphal branching than the parent cell. A mutant cell having a“more extensive hyphal branching” is defined herein as a mutant cellhaving a hyphal growth unit length which is at least 10% less than thehyphal growth unit length of the parent cell. Preferably, the hyphalbranching of the mutant cell is at least about 20% more branched, andmore preferably at least about 30% more branched than the parent cell.Measurement of the hyphal growth unit length may be made according tothe method of Trinci et al., 1973, Archiv für Mikrobiologie 91: 127-136.One way of making this determination is to measure the average distancebetween branches in fungal hyphae (see, for example, Withers et al.,1994, Mycological Research 98: 95-100).

[0030] The mutant cells of the present invention also have an improvedproperty for production of a heterologous polypeptide than the parentcell, when the mutant and parent cells are cultured under the sameconditions. The mutants obtained by the methods of the present inventionmay possess improved growth characteristics in fermentation where themorphology gives rise to lower viscosity in the fermenter, in turnleading to easier mixing, better aeration, better growth, andultimately, enhanced yield of heterologous polypeptide produced by themutant strain relative to the parent strain. In a preferred embodiment,the improved property is selected from the group consisting of(a)increased yield of the heterologous polypeptide, (b) improved growth,(c) lower viscosity, and (d) better secretion. In a most preferredembodiment, the improved property is increased yield of the heterologouspolypeptide. In another most preferred embodiment, the improved propertyis improved growth. In another most preferred embodiment, the improvedproperty is lower viscosity. In another most preferred embodiment, theimproved property is better secretion.

[0031] In order to determine whether a mutant cell has an improvedproperty for production of a heterologous polypeptide than the parentcell, a nucleic acid construct comprising a nucleic acid sequenceencoding the heterologous polypeptide of interest is introduced intoboth the parent strain and the morphological mutant, e.g., bytransformation. “Nucleic acid construct” is defined herein as a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acid which are combined and juxtaposed in a mannerwhich would not otherwise exist in nature. The mutant cell is preferablytransformed with a vector comprising the nucleic acid construct followedby integration of the vector into the host chromosome. “Transformation”means introducing a nucleic acid construct into a host cell so that theconstruct is maintained as a chromosomal integrant. Integration isgenerally considered to be an advantage as the nucleic acid sequenceencoding the heterologous polypeptide is more likely to be stablymaintained in the cell. Integration of the vector into the hostchromosome occurs by homologous or non-homologous recombination.Transformation is achieved using those techniques adapted for the fungalhost being used, many of which are well known in the art. Suitableprocedures for transformation of Aspergillus cells are described in EP238 023, Christensen et al., 1988, Bio/Technology 6:1419-1422, andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81:1470-1474. A suitable method of transforming Fusarium species isdescribed by Malardier et al., 1989, Gene 78:147-156 or in copendingU.S. Ser. No. 08/269,449.

[0032] After transformation of the mutant and the parent cell with avector containing a gene encoding a heterologous polypeptide, sporesgathered from the mutants and parent are used to inoculate liquidmedium. After a suitable period of growth, supernatants are tested foractivity of the polypeptide.

[0033] When the improved property is yield, the levels of expression ofthe heterologous polypeptide are compared between the mutant and parentstrains. In such case, the productive phase of the mutant's fermentationshould be extended. In a preferred embodiment, the morphological mutantproduces at least about 10% more heterologous polypeptide than theparent strain when each strain is cultured under identical conditions.More preferably, the mutant produces at least 20%, and most preferablyat least 30%, more heterologous polypeptide. In some cases, the mutantmay produce as much as 50%-100% more polypeptide, or even higher. Sincein all cultures it is expected that a range of expression levels maybeobserved, it is understood that this figure can represent the mean,median or maximum level of expression in a population of transformantstrains.

[0034] When the improved property is reduced viscosity, viscosity can bedetermined by any means known in the art, e.g., Brookfield rotationalviscometry (defined or unlimited shear distance and any type of spindleconfiguration), kinematic viscosity tubes (flow-through tubes), fallingball viscometer or cup-type viscometer. The preferred host cells of thepresent invention exhibit about 90% or less of the viscosity levelproduced by an the parent cell under identical fermentation conditions,preferably about 80% or less, and more preferably about 50% or less.

[0035] The present morphological mutants can be used to express anyprokaryotic or eukaryotic heterologous peptide or polypeptide ofinterest, and are preferably used to express eukaryotic peptides orpolypeptides. Of particular interest for these species is their use inexpression of heterologous polypeptides, in particular fungalpolypeptides, especially fungal enzymes. The morphological mutants canbe used to express enzymes such as a hydrolase, an oxidoreductase, anisomerase, a ligase, a lyase, or a transferase. More preferably, theenzyme is an aminopeptidase, an amylase, a carboxypeptidase, a catalase,a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an esterase, a glucoamylase, analpha-galactosidase, a beta-galactosidase, an alpha-glucosidase,beta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, amannosidase, a mutanase, an oxidase, a pectinolytic enzyme, aperoxidase, a phenoloxidase, phytase, a proteolytic enzyme, aribonuclease, a xylanase, or a xylose isomerase. It will be understoodby those skilled in the art that the term “fungal enzymes” includes notonly native fungal enzymes, but also those fungal enzymes which havebeen modified by amino acid substitutions, deletions, additions, orother modifications which may be made to enhance activity,thermostability, pH tolerance and the like. Other polypeptides that canbe expressed include, but are not limited to, mammalian polypeptidessuch as insulin, insulin variants, receptor proteins and portionsthereof, and antibodies and portions thereof.

[0036] The mutants may also be used in recombinant production ofpolypeptides which are native to the host cells. Examples of such useinclude, but are not limited to, placing a gene encoding the polypeptideunder the control of a different promoter to enhance expression of thepolypeptide, to expedite export of a native polypeptide of interestoutside the cell by use of a signal sequence, or to increase the copynumber of a gene encoding the protein normally produced by the subjecthost cells. Thus, the present invention also encompasses, within thescope of the term “heterologous polypeptide”, such recombinantproduction of homologous polypeptides, to the extent that suchexpression involves the use of genetic elements not native to the hostcell, or use of native elements which have been manipulated to functionin a manner not normally seen in the host cell.

[0037] In the present invention, the nucleic acid construct is operablylinked to one or more control sequences capable of directing theexpression of the coding sequence in the mutant cell under conditionscompatible with the control sequences. The term “coding sequence” asdefined herein is a sequence which is transcribed into mRNA andtranslated into a polypeptide of the present invention when placed underthe control of the control sequences. The boundaries of the codingsequence are generally determined by a translation start codon ATG atthe 5′-terminus and a translation stop codon at the 3′-terminus. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

[0038] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for expression of thecoding sequence of the nucleic acid sequence. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, a polyadenylation sequence, a propeptide sequence, a promoter, asignal sequence, and a transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

[0039] The control sequence may be an appropriate promoter sequence, anucleic acid sequence which is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscription and translation control sequences which mediate theexpression of the polypeptide. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceand may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalcell are promoters obtained from the genes encoding Aspergillus oryzaeTAKA amylase (as described in U.S. patent application Ser. No.08/208,092, the contents of which are incorporated herein by reference),Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillusniger or Aspergillus awamori glucoamylase (glaA), Rhizomucor mieheilipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumoxysporum trypsin-like protease (as described in U.S. Pat. No.4,288,627, which is incorporated herein by reference), and hybridsthereof. Particularly preferred promoters for use in filamentous fungalcells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from thegenes encoding Aspergillus niger neutral α-amylase and Aspergillusoryzae triose phosphate isomerase), and glaA promoters.

[0040] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. Preferred terminators for filamentous fungalcells are obtained from the genes encoding Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

[0041] The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencewhich is functional in the host cell of choice may be used in thepresent invention. Preferred leaders for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase andAspergillus oryzae triose phosphate isomerase.

[0042] The control sequence may also be a polyadenylation sequence, asequence which is operably linked to the 3′ terminus of the nucleic acidsequence and which, when transcribed, is recognized by the host cell asa signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence which is functional in the host cell of choicemay be used in the present invention. Preferred polyadenylationsequences for filamentous fungal host cells are obtained from the genesencoding Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, andAspergillus niger alpha-glucosidase.

[0043] The control sequence may also be a signal peptide coding region,which codes for an amino acid sequence linked to the amino terminus ofthe polypeptide which can direct the expressed polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region which encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion which is foreign to that portion of the coding sequence whichencodes the secreted polypeptide. The foreign signal peptide codingregion may be required where the coding sequence does not normallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to obtain enhanced secretion of thepolypeptide relative to the natural signal peptide coding regionnormally associated with the coding sequence. The signal peptide codingregion may be obtained from a glucoamylase or an amylase gene from anAspergillus species, a lipase or proteinase gene from a Rhizomucorspecies, the gene for the alpha-factor from Saccharomyces cerevisiae, anamylase or a protease gene from a Bacillus species, or the calfpreprochymosin gene. However, any signal peptide coding region capableof directing the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention. An effectivesignal peptide coding region for filamentous fungal host cells is thesignal peptide coding region obtained from Aspergillus oryzae TAKAamylase gene, Aspergillus niger neutral amylase gene, the Rhizomucormiehei aspartic proteinase gene, the Humicola lanuginosa cellulase gene,or the Rhizomucor miehei lipase gene.

[0044] The control sequence may also be a propeptide coding region,which codes for an amino acid sequence positioned at the amino terminusof a polypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

[0045] The vector may be any vector which can be conveniently subjectedto recombinant DNA procedures and can bring about the expression of thenucleic acid sequence encoding the polyppetide. The choice of the vectorwill typically depend on the compatibility of the vector with the hostcell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector system may be a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell.The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genome.For integration, the vector may rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector for stableintegration of the vector into the genome by homologous or nonhomologousrecombination. Alternatively, the vector may contain additional nucleicacid sequences for directing integration by homologous recombinationinto the genome of the host cell. The additional nucleic acid sequencesenable the vector to be integrated into the host cell genome at aprecise location(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to1,500 base pairs, preferably 400 to 1,500 base pairs, and mostpreferably 800 to 1,500 base pairs, which are highly homologous with thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleic acid sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

[0046] The vectors preferably contain one or more selectable markerswhich permit easy selection of transformed cells. A selectable marker isa gene the product of which provides for biocide resistance, resistanceto heavy metals, prototrophy to auxotrophs, and the like. A selectablemarker for use in a filamentous fungal host cell may be selected fromthe group including, but not limited to, amdS(acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), trpC (anthranilate synthase), and glufosinateresistance markers, as well as equivalents from other species. Preferredfor use in an Aspergillus cell are the amdS and pyrG markers ofAspergillus nidulans or Aspergillus oryzae and the bar marker ofStreptomyces hygroscopicus. Furthermore, selection may be accomplishedby co-transformation, e.g., as described in WO 91/17243, where theselectable marker is on a separate vector.

[0047] According to a preferred embodiment of the present invention, thehost is transformed with a single DNA vector including both theselection marker and the remaining heterologous DNA to be introduced,including promoter, the gene for the desired polypeptide andtranscription terminator and polyadenylation sequences.

[0048] The procedures used to ligate the elements described above toconstruct the nucleic acid constructs and vectors are well known to oneskilled in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NewYork), 1989, supra).

[0049] The present invention also relates to methods of producing aheterologous polypeptide, comprising: (a) cultivating a mutant cell ofthe present invention which comprises a nucleic acid sequence encodingthe heterologous polypeptide; and (b) recovering the heterologouspolypeptide.

[0050] The cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art (see,e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L.,editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).Suitable media are available from commercial suppliers or may beprepared according to published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the polypeptide is secreted intothe nutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it is recovered from celllysates.

[0051] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide. Procedures fordetermining enzyme activity are well known in the art.

[0052] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The recovered polypeptide may then be further purified bya variety of chromatographic procedures, e.g., ion exchangechromatography, gel filtration chromatography, affinity chromatography,or the like.

[0053] The polypeptides of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

EXAMPLES

[0054] Strains and Media

[0055] The starting strains are alpha-amylase deficient, pyrG-negativeAspergillus oryzae HowB425 and Fusarium A3/5. Morphological mutants ofFusarium A3/5 designated CC1-3, CC2-3, and MC3-5 (Wiebe et al., 1992,Mycological Research 96: 555-562; Wiebe et al., 1991, MycologicalResearch 95: 1284-1288; Wiebe et al., 1991, Mycological Research 96:555-562) are highly branched, colonial variants.

[0056] PDA plates contain 39 g/l Potato Dextrose Agar (Difco) and aresupplemented with 10 mM uridine for pyrG auxotrophs unless otherwiseindicated.

[0057] MY50N medium is comprised of 62.5 g of Nutriose, 2.0 g ofMgSO₄-7H₂O, 2.0 g of KH₂PO₄, 4.0 g of citric acid, 8.0 g of yeastextract, 2.0 g of urea, 0.1 g of CaCl₂, and 0.5 ml of trace metalssolution pH 6.0 per liter. MY50N shake-flask medium is diluted 1:100with glass distilled water for use in microtiter growth experiments(MY50N/100). Cultures are grown at a temperature between 28-37° C.

[0058] Minimal medium plates are comprised of 6.0 g of NaNO₃, 0.52 g ofKCl, 1.52 g of KH₂PO₄, 1.0 ml of trace metals solution, 20 g of NobelAgar (Difco), 20 ml of 50% glucose, 20 ml of methionine (50 g/l), 20 mlof biotin (200 mg/l), 2.5 ml of 20% MgSO₄-7H₂O, and 1.0 ml of mg/mlstreptomycin per liter. The agar medium is adjusted to pH 6.5 prior toautoclaving and then glucose, methionine, biotin, MgSO₄-7H₂O, andstreptomycin are added as sterile solutions to the cooled autoclavedmedium and poured into plates.

[0059] The trace metals solution (1000×) is comprised of 22 g ofZnSO₄-7H₂O, 11 g of H₃BO₃, 5 g of MnCl₂-4H₂O, 5 g of FeSO₄-7H₂O, 1.6 gof CoCl₂-5H₂O, 1.6 g of (NH₄)₆MO₇O₂₄, and 50 g of Na₄EDTA per liter.

[0060] COVE plates are comprised of 343.3 g of sucrose, 20 ml of COVEsalts solution, 10 ml of 1 M acetamide, 10 ml of 3 M CsCl, and 25 g ofNobel agar per liter. The COVE salts (50×) solution is comprised of 26 gof KCl, 26 g of MgSO₄-7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE tracemetals solution. COVE trace metals solution is comprised of 0.04 g ofNaB₄O₇-10H₂O, 0.040 g of CuSO₄-5H₂O, 0.70 g of FeSO₄-H₂O, 0.80 g ofNa₂MoO₂-2H₂O, and 10 g of ZnSO₄ per liter.

[0061] M400Da medium is comprised of 50 g of maltodextrin, 2.0 g ofMgSO₄-7H₂O, 2.0 g of KH₂PO₄, 4.0 g of citric acid, 8.0 g of yeastextract, 2.0 g of urea, and 0.5 ml of trace metals solution per liter.The medium is adjusted to pH 6.0 with 5 N NaOH. The trace metalssolution is comprised of 14.3 g of ZnSO₄-7H₂O, 2.5 g of CuSO₄-5H₂O, 0.5g of NiCl₂-6H₂O, 13.8 g of FeSO₄-7H₂O, 8.5 g of MnSO₄-H₂O, and 3.0 g ofcitric acid per liter.

Example 1

[0062] Mutagenesis of Aspergillus oryzae strain HowB425

[0063]Aspergillus oryzae strain HowB425 spores are harvested from solidmedium and suspended to a concentration of 2.2×10⁷/ml in 0.01% Tween 80.Five ml of spore suspension are pipetted into a 90 mm plastic petri dishand the spores are irradiated for one minute with ultraviolet light toapproximately 5% survival. The mutagenized spores are kept in the darkfor one hour and then plated to PDA+50 mg/l uridine plates.

[0064] The frequencies of spore color mutants obtained from thismutagenesis treatment are 8.8×10⁻⁵ for white and 5.9×10⁻⁵ for yellowspored mutants. A total of about 34,000 viable colonies (250 to 800 perplate) are screened visually for a restricted colonial phenotype.Eighty-eight restricted colonials are selected. The edges of therestricted colonies growing on the plate are examined under a microscope(200×) for a high mycelial branching phenotype. Thirty-six of theselected colonials are selected as having a more extensive hyphalbranching pattern than the Aspergillus oryzae HowB425 control strain andare purified by restreaking spores onto PDA+uridine plates. After growthand sporulation, the strains are repurified in a similar fashion. The 36mutants are re-examined for the colonial and high-branching phenotypes.Twelve of the 36 retested positive in both assays and are selected forheterologous polypeptide expression analysis. The frequency of mutantsrecovered is 12/34,000 or about 3.5×10⁻⁴. The colonial mutants areclassified by examination of their hyphal branching phenotypes (TableI). The colony morphology of the control strain and one of the mutantson PDA+uridine solid medium are shown in FIG. 2. TABLE I Phenotypes ofMorphological Mutants Phenotypes Strains Wild Type growth and lowbranching HowB425 (control) Colonial growth and medium branching JeRS306JeRS307 JeRS314 JeRS316 JeRS318 Colonial growth and high branchingJeRS303 JeRS304 JeRS315 JeRS320 Colonial growth and very high branchingJeRS313 Colonial growth and highly branched short hyphae JeRS317Colonial growth and highly branched very short hyphae JeRS305

Example 2

[0065] Lipase Expression Plasmid

[0066] A map of the lipase expression plasmid pJeRS23 is shown inFIG. 1. pJeRS23 contains the amdS gene from Aspergillus nidulans frombases −118 to 2191 (relative to the ATG start codon), thepTAKA-TPI/Lipolase/AMGt lipase expression cassette from pMHan37, theAspergillus oryzae pyrG gene, and pUC19 sequences.

Example 3

[0067]Aspergillus oryzae Transformation

[0068] Cultures to be transformed are grown in 20 ml of 1% yeastextract-2% Peptone (Difco)-2.5% glucose at 37° C. for 16-20 hours withagitation. Each culture is mixed with 10 ml of 1.2 M MgSO₄, and themycelia are recovered by filtration on Miracloth (CalBiochem, La Jolla,Calif.) or by centrifugation, washed with 1.2 M MgSO₄, and thenresuspended in 10 ml of 5 mg/ml NOVOZYM 234 (Novo Nordisk A/S,Bagsvaerd, Denmark) in 1.2 M MgSO₄. The suspension is incubated withgentle agitation for approximately one hour at 37° C. to generateprotoplasts. Undigested mycelia are removed by filtration through alayer of sterile Miracloth. Protoplasts are recovered by centrifugationat 3600× g. They are then washed with 10 ml of ST (1 M sorbitol-10 mMTris pH 7.5), centrifuged, washed with 10 ml of STC (1 M sorbitol-10 mMTris pH 7.5-10 mM CaCl₂), centrifuged, and then resuspended in 1.0 ml ofSTC. The concentration of protoplasts is determined and the finalconcentration is adjusted to between 2×10⁶ and 1×10⁷/ml with STC. Analiquot of 0.1 ml protoplasts is mixed with 5 μl of pJeRS23 DNA (about 5μg) in a Falcon 2059 polypropylene tube and incubated at roomtemperature for 20 minutes. One ml of SPTC (0.8 M sorbitol-40%polyethylene glycol 4000-50 mM CaCl₂-50 mM Tris pH 8) is added and thesuspension is mixed with gentle shaking. The suspension is incubated atroom temperature for 20 minutes and then 7 ml of molten overlay agar (1×COVE salts, 0.8 M sucrose, 1% low melt agarose) is added and thesuspension is poured onto a COVE plate. The plates are incubated at 37°C.

Example 4

[0069] Lipase Assay

[0070] Assay substrate is prepared by diluting 1:5 the stock substrate(10 μl of p-nitrophenylbutyrate/ml DMSO) into MC buffer (3 mM CaCl₂-0.1MMOPS pH 7.5) immediately before use. Standard Lipolase® contains 1000LU/ml of 50% glycerol-0.66 mM CaCl₂-33 mM Tris pH 7.5 and is stored at−20° C. Standard Lipolase® is diluted 1/100 in MC buffer just beforeuse. Broth samples are diluted in MC buffer and 100 μl aliquots of thediluted broth samples are pipetted into 96-well microtiter dishesfollowed by 100 μl of diluted substrate. The absorbance at nm isrecorded as a function of time. Broth lipase units/ml (LU/ml) arecalculated relative to a Lipolase® standard.

Example 5

[0071] Lipolase® Expression

[0072] Each of the twelve mutants is transformed with the Lipolase®expression plasmid pJeRS23 described in Example 2 and the transformantsare selected by their prototrophy for uridine and ability to grow usingacetamide as sole nitrogen source. A parallel transformation isperformed with the parent strain Aspergillus oryzae HowB425. Theconidiated transformants are restreaked once to COVE plates and sporesfrom individual colonies are used to inoculate a 90 mm COVE plate. Aftersporulation, the spores are harvested in 0.01% Tween 80. A 10 μl aliquotof each spore suspension is used to inoculate a well in a 24-wellmicrotiter plate that contains 1 ml of MY50N/100 liquid medium.Experiments are started on two different days (Experiments A and B) withthe entire set of Aspergillus oryzae HowB425 control transformantsincluded each day. The microtiter plates are grown for 3-5 days at 37°C., 100 rpm agitation, and the culture supernatants are assayed forlipase activity as described in Example 4.

[0073] The results are shown in Table II. A graphic representation oftwo of the mutants is shown in FIGS. 3 and 4. Although it is typicalthat individual transformants obtained following the introduction of DNAexpression plasmids into any given Aspergillus oryzae host will vary intheir ability to produce and secrete a heterologous polypeptide, and thenumber of transformants in each mutant strain is fairly small, theexpression profiles for mutants JeRS316 (FIG. 3) and JeRS317 (FIG. 4)appear to be shifted further to higher lipase expression values ascompared with the control. TABLE II Lipolase ® Expression inMorphological Mutants Standard of number of Mean Deviation Median MaxStrain transformants (LU/ml) (LU/ml) (LU/ml) (LU/ml) Experiment AHowB425 12 6.49 3.28 5.47 12.10 JeRS305 8 2.11 2.64 0.62 6.61 JeRS306 127.44 5.49 6.57 15.40 JeRS307 10 8.31 6.49 6.74 24.00 JeRS313 5 4.54 3.842.40 8.98 JeRS315 6 8.96 8.60 7.27 25.80 JeRS317 9 12.20 6.85 10.9728.00 JeRS318 9 1.53 3.68 0.00 11.20 JeRS320 9 9.47 7.97 5.11 22.56Experiment B HowB425 12 12.10 4.04 11.40 20.00 JeRS303 12 7.51 9.55 4.3431.70 JeRS304 10 12.90 11.30 9.60 36.50 JeRS314 12 11.40 5.89 12.2021.60 JeRS316 12 18.00 15.00 13.40 44.50

Example 6

[0074] Fermentation of Aspergillus oryzae mutant JeRS316

[0075] To determine if the morphology mutants exhibit a superiorfermentation behavior in comparison with the parent wild type morphologystrain, one transformant each of the parent strain Aspergillus oryzaeHowB425 and the mutant strain JeRS316 transformed with plasmid pJeRS23are grown in a tank fermenter under fermentation conditions.

[0076] The morphology of the control culture at the end of thefermentation is typical for Aspergillus oryzae grown under theseconditions. The culture is very viscous with a thick and grainy slowmixing appearance. Large air bubbles are visible in the tank. Incontrast, the JeRS316 transformant displays a low viscosity,filamentous, easy mixing morphology with a small degree of pelletformation throughout the fermentation. Large air bubbles are notroutinely observed in the tank. The expression of the heterologouslipase is examined and the results are shown in FIG. 5. If the mutant issuperior to the parent in fermentation, the expectation is that theproductive phase of the fermentation would be extended for the mutant.The results are reported as the ratio of [lipase titer in the culturebroth at time (x)]/[lipase titer at time (42 hours)]. This analysisnormalizes the expression data for the fact that not all transformantsare equivalent in their absolute level of expression. As predicted foran improved morphology mutant, the heterologous polypeptide productivephase of the fermentation is extended significantly in strain JeRS316 ascompared with the control. The final expression of the lipase in thebroth of the morphology mutant culture is about five times higher intiter than the control.

Example 7

[0077] Construction of Fusarium expression Vector pJRoy30

[0078] The EcoRV site at −15 in the Fusarium oxysporum trypsin genepromoter ofpJRoy20 (Royer et al., 1995, Bio/Technology 13: 1479-1483)and the NcoI site present at +243 in the CAREZYME™ (Novo Nordisk A/S,Bagsværd, Denmark) cellulase coding region are utilized to create anexact fusion between the Fusarium oxysporum trypsin gene promoter andthe CAREZYME™ cellulase gene. A PCR fragment containing −18 to −1 of theFusarium oxysporum trypsin gene promoter directly followed by −1 to +294of the CAREZYME™ cellulase gene is generated from the CAREZYME™ vectorpCaHj418 (see FIG. 6) using the following primers: FORWARD EcoRV 5′ctcttggatatctatctcttcaccATGCGTTCCTCCCCCCTCCT 3′ (SEQ ID NO: 1) REVERSE5′ CAATAGAGGTGGCAGCAAAA 3′ (SEQ ID NO: 2)

[0079] Lower case letters in the forward primer are bp −24 to −1 of theFusarium oxysporum trypsin gene promoter, while upper case letters arebp 1 to 20 of CAREZYME™.

[0080] The PCR conditions used are 95° C. for 5 minutes followed by 30cycles each at 95° C. for 30 seconds, 50° C. for 1 minute, and 72° C.for 1 minute. The resulting 0.32 kb fragment is cloned into vector pCRIIusing Invitrogen's TA Cloning Kit (Invitrogen, La Jolla, Calif.)resulting in pDM148 (see FIG. 7). The 0.26 kb EcoRV/NcoI fragment isisolated from pDM148 and ligated to the 0.69 kb NcoI/BglII fragment frompCaHj418 and cloned into EcoRV/BamHI digested pJRoy20 to create pDM149(see FIG. 8).

[0081] pMT1612 is constructed by introducing a 575 bpBamH1 BamH1fragment containing the bar gene from pBIT (Straubinger et al., 1992,Fungal Genetics Newsletter 39:82-83) into pIC19H (Marsh et al., 1984,Gene 32:481-485) cut with BamHI/BglII. The bar gene is then isolated asa BamH1-XhoI fragment and inserted into BamHI-XhoI cut pJaL154 (FIG. 9)to generate pMT1612 (FIG. 10). The 3.2 kb EcoR1 CAREZYME™ cellulaseexpression cassette is transferred from pDM149 into EcoR1 cut bastamarker pMT1612 to generate pJRoy30 (FIG. 11).

Example 8

[0082] Transformation of Fusarium

[0083]Fusarium strain A3/5 (ATCC 20334) and Fusarium strain A3/5 highlybranched morphological mutants CC1-3, CC1-8, CC2-3, and MC3-5 (Wiebe etal., 1992, Mycological Research 96:555-562) are grown on 10×15 mm petriplates of Vogels medium (Vogel, 1964, Am. Nature 98:435-446) plus 1.5%glucose and agar for 3 weeks at 25° C. Conidia (approximately 10⁸ perplate) are dislodged in 10 ml of sterile water using a transfer loop andpurified by filtration through 4 layers of cheesecloth and finallythrough one layer of Miracloth. Conidial suspensions are concentrated bycentrifugation. Fifty ml of YPG medium comprised of 1% yeast extract, 2%bactopeptone, and 2% glucose are inoculated with 10⁸ conidia, andincubated for 14 hours at 24° C., 150 rpm. Resulting hyphae are trappedon a sterile 0.4 μm filter and washed successively with steriledistilled water and 1.0 M MgSO₄. The hyphae are resuspended in 10 ml ofNOVOZYM ₂₃₄™ solution (2-10 mg/ml in 1.0 M MgSO₄) and digested for 15-30minutes at 34° C. with agitation at 80 rpm. Undigested hyphal materialis removed from the resulting protoplast suspension by successivefiltration through 4 layers of cheesecloth and through Miracloth. Twentyml of 1 M sorbitol are passed through the cheesecloth and Miracloth andcombined with the protoplast solution. After mixing, protoplasts(approximately 5×10⁸) are pelleted by centrifugation and washedsuccessively by resuspension and centrifugation in 20 ml of 1 M sorbitoland in 20 ml of STC. The washed protoplasts are resuspended in 4 partsSTC and 1 part SPTC at a concentration of 1-2×10⁸/ml. One hundred μl ofprotoplast suspension are added to 5 μg pJRoy30 and 5 μl heparin (5mg/ml in STC) in polypropylene tubes (17×100 mm) and incubated on icefor 30 minutes. One ml of SPTC is mixed gently into the protoplastsuspension and incubation is continued at room temperature for 20minutes. Twenty five ml of molten solution (cooled to 40° C.) consistingof COVE salts, 25 mM NaNO₃, 0.8 M sucrose and 1% low melting agarose(Sigma Chemical Company, St. Louis, Mo.) are mixed with the protoplastsand then plated onto an empty 150 mm petri plate. Incubation iscontinued at room temperature for 10 to 14 days. After incubation atroom temperature for 24 hours, 25 ml of the identical medium plus basta(5 mg/ml) are overlayed onto the petri plate. Basta is obtained fromAgrEvo (Hoechst Schering, Rodovre, Denmark) and is extracted twice withphenol:chloroform:isoamyl alcohol (25:24:1), and once withchloroform:isoamyl alcohol (24:1) before use.

Example 9

[0084] Expression of Cellulase Activity

[0085] Transformants of Fusarium A3/5, CC1-3, CC2-3, and MC3-5 arecultured on M400Da medium in microtiter plates and shake flasks for 7days at 37° C. One transformant each of Fusarium A3/5, CC 1-3, CC2-3,and MC3-5 are cultivated in fermentors under suitable fermentationconditions in a medium containing typical carbon and nitrogen sources aswell as mineral salts and trace metals.

[0086] Cellulase activity is measured using the following procedure.Volumes of 5 μl of various dilutions of a cellulase standard and 1-5 μlof samples are pipetted into a 96-well plate. The cellulase standard(CAREZYME™, Novo Nordisk A/S, Bagsverd, Denmark) is diluted to 150, 100,50, 25, 12.5 and 6.25 ECU/ml in 100 mM MOPS pH 7.0. The substrate isprepared by dissolving azo-carboxymethylcellulose (Azo-CMC) at 2% w/v in100 mM MOPS pH 7.0 and stirring at 80° C. for 10 minutes. A volume of 65microliters of the azo-CMC substrate solution is pipetted into each ofthe sample wells and mixed. The 96-well plate is incubated in a waterbath at 45° C. for 30 minutes and is then placed on ice for 2 minutes. Avolume of 175 microliters of stop reagent is added to each well andmixed well. The stop reagent is prepared by suspending 0.2 g of ZnCl₂ in20 ml of 0.25 M MOPS pH 7.0 and adding the suspension to 80 ml ofacidified ethanol containing 1.1 ml of concentrated HCl per liter ofethanol. The 96-well plate is centrifuged at 3000 rpm for 10 minutes ina Sorval RT 6000B centrifuge. After centrifugation is complete, 50 μl ofeach supernatant is transferred to a new 96-well plate containing 50 μlof water per well. The absorbance at 600 nm is measured.

[0087] The results for the microtiter plate, shake flask, and fermentorcultures are presented in Table III where the maximum cellulase yield isnormalized to 1.0. In microtiter plate culture, transformants of CC2-3and MC3-5 produce levels of cellulase which are 22% and 46% higher,respectively, compared to the parent strain. In shake flask culture,transformants of CC 1 -3, CC2-3 5, and MC3-5 produce levels of cellulasewhich are 85%, 54%, and 7% higher, respectively, compared to the parentstrain. In fermentors, of CC1-3, CC2-3 5, and MC3-5 produce levels ofcellulase which are 136%, 3%, and 8% higher, respectively, compared tothe parent strain. TABLE III Production of cellulase by transformants ofthe wild type strain (A3/5) and morphological mutants (CC1-3, CC2-3, andMC3-5). Microtiter Plate Shake Flask Fermentor Host Maximum° n* Maximumn Maximum n A3/5 1.0  5 1.0  6 1.0  1 CC1-3 0.29 4 1.85 2 2.36 1 CC2-31.22 1 1.54 1 1.03 1 MC3-5 1.46 2 1.07 2 1.08 1

[0088]

1 2 1 44 DNA Fusarium oxysporum 1 ctcttggata tctatctctt caccatgcgttcctcccccc tcct 44 2 20 DNA Fusarium oxysporum 2 caatagaggt ggcagcaaaa20

What is claimed is:
 1. A method for obtaining a mutant cell whichproduces a heterologous protein, comprising: (a) producing a firstpopulation of presumptive mutant cells from a Humicola, Mucor,Myceliophthora, Scytalidium, Thielavia, or Tolypocladium parent cell;(b) identifying from the first population a second population ofpresumptive mutant cells having a more restricted colonial phenotype ora more extensive hyphal branching than the parent cell; and (c)identifying from the second population the mutant cell, comprising anucleic acid sequence encoding a heterologous polypeptide, which has aradial extension rate which is at least 10% less than the parent cell,has a hyphal growth unit length that is at least 10% less than theparent cell, and has one or more improved properties selected from thegroup consisting of (i) produces at least about 10% more heterologouspolypeptide than the parent cell, (ii) exhibits about 90% or less of theviscosity of the parent cell, and (iii) secretes more heterologousprotein than the parent cell, when cultivated under the same conditions;and (d) obtaining the mutant cell.
 2. The method of claim 1, wherein instep (b), the second population of presumptive mutant cells has a morerestricted colonial phenotype and a more extensive hyphal branching thanthe parent cell.
 3. The method of claim 1, wherein the radial extensionrate is at least 20% less than the parent cell.
 4. The method of claim1, wherein the hyphal growth unit length is at least 20% less than theparent cell.
 5. The method of claim 1, wherein the mutant cell producesat least about 10% more heterologous polypeptide than the parent cell.6. The method of claim 1, wherein the mutant cell exhibits about 90% orless of the viscosity of the parent cell.
 7. The method of claim 1,wherein the mutant cell has a further improved property of an improvedgrowth.
 8. A method for producing a heterologous polypeptide,comprising: (a) cultivating a mutant cell of a Humicola, Mucor,Myceliophthora, Scytalidium, Thielavia, or Tolypocladium parent cellunder conditions suitable for production of the heterologouspolypeptide, wherein the mutant cell comprises a nucleic acid sequenceencoding the heterologous polypeptide, and wherein the mutant cell has aradial extension rate which is at least 10% less than the parent cell,has a hyphal growth unit length that is at least 10% less than theparent cell, and has one or more improved properties selected from thegroup consisting of (i) produces at least about 10% more heterologouspolypeptide than the parent cell, (ii) exhibits about 90% or less of theviscosity of the parent cell, and (iii) secretes more heterologousprotein than the parent cell, when cultivated under the same conditions;and (b) recovering the heterologous polypeptide.
 9. The method of claim8, wherein the radial extension rate is at least 20% less than theparent cell.
 10. The method of claim 8, wherein the hyphal growth unitlength is at least 20% less than the parent cell.
 11. The method ofclaim 8, wherein the mutant cell produces at least about 10% moreheterologous polypeptide than the parent cell.
 12. The method of claim8, wherein the mutant cell exhibits about 90% or less of the viscosityof the parent cell.
 13. The method of claim 8, wherein the mutant cellhas a further improved property of an improved growth.
 14. A mutant cellof a Humicola, Mucor, Myceliophthora, Scytalidium, Thielavia, orTolypocladium parent cell comprising a nucleic acid sequence encoding aheterologous polypeptide, wherein the mutant cell has a radial extensionrate which is at least 10% less than the parent cell, has a hyphalgrowth unit length that is at least 10% less than the parent cell, andhas one or more improved properties selected from the group consistingof (i) produces at least about 10% more heterologous polypeptide thanthe parent cell, (ii) exhibits about 90% or less of the viscosity of theparent cell, and (iii) secretes more heterologous protein than theparent cell, when cultivated under the same conditions.
 15. The mutantcell of claim 14, wherein the radial extension rate is at least 20% lessthan the parent cell.
 16. The mutant cell of claim 14, wherein thehyphal growth unit length is at least 20% less than the parent cell. 17.The mutant cell of claim 14, wherein the mutant cell produces at leastabout 10% more heterologous polypeptide than the parent cell.
 18. Themutant cell of claim 14, wherein the mutant cell exhibits about 90% orless of the viscosity of the parent cell.
 19. The method of claim 14,wherein the mutant cell has a further improved property of an improvedgrowth.